Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
WO94/00164 PCT/GB93/01350
~ 2134987
,_
8U~TAINED P~l~'~E ALGINATE FIBRB AND PP~r~~ FOR THE
PR~PARATION ~n~K~
s The present invention relates to a medicament--
impregnated alginate fibre. More particularly, the
invention concerns a fibre from which medicament can be
released in a controlled manner, and to the application
of this fibre in the preparation of alginate fabrics and
wound dressings.
A number of methods for producing conventional
alginate fibres are described in the art. The extrusion
of alginate solutions into an aqueous solution containing
calcium ions to form yarns of calcium alginate filaments
is known, for example, from British Patents Nos. 567641,
568177, 571657 and 624987. Such fabrics were generally
prepared by knitting a yarn of calcium alginate filaments
and partially converting the calcium alginate to the
sodium form to form a calcium/sodium alginate containing,
for example, 30-70 percent by weight of the carboxyl
groups of the alginate in the calcium form.
US Patent No. 4421583 discloses a process for
making a non-woven alginate fabric useful as a wound
dressing which comprises spreading a tow of calcium
alginate filaments in a flow of water; overfeeding the
spread filaments onto a water-pervious support so that
the filaments cross over each other; and drying the
filaments so that the filaments become bonded to each
other at their points of contact where they cross over
each other. Non-woven alginate fabrics having this high
calcium content of 70 to 95 percent are said to be useful
for example as throat swabs, which after swabbing are
dissolved in sodium hexametaphosphate solution. The non-
woven alginate fabrics can be treated such that calcium
WO94/00164 PCT/GB93/01350
213~987 _
is partially replaced by sodium, for example by treatment
with alcoholic sodium hydroxide and sodium acetate so
that from 30 to 70 percent of the carboxyl ~LoU~ are in
the sodium form. The sodium calcium alginate fabrics
produced are said to be useful as haemostatic wound
dressings which can be absorbed by body fluids, for use
for example in deep surgery or after tooth extraction.
W0-A-84/03705 describes a method of producing
alginate fibres for fabric of increased solubility in
water or saline solutions for application in the medical
field and in particular for haemostatic dressings.
US Patent No. 4562110 describes a method of
producing dried alginate fibre material which comprises
spinning an alginate solution into an aqueous salt bath.
The wet-spun continuous fibres of insoluble alginate, in
which the fibres are separate but generally parallel, are
collected, and the sheet of fibres is laid on a moving
conveyor belt which is travelling at a slower speed than
the sheet fibre feed speed, so that the fibres become
overlaid in a web of defined width. The fibre web is
dried and stretched in a manner to reduce the bonding
between the fibres. The web is said to be useful as a
component of medicated and surgical dres~sings.
Wound dressings which may incorporate certain
medicaments are also known in the art. For example, EP-
A-0279118 describes an adhesive product suitable for use
on wounds comprising a backing layer of a moisture vapour
transmitting continuous polymer film and a layer of a
pressure sensitive adhesive containing inter alia at
least 30% by weight of alginate. The adhesive product
described therein can contain a medicament, such as a
chlorhexidine derivative, normally in the adhesive layer.
Alternatively, the medicament may form part of the
alginate polymer used for the pressure sensitive adhesive
-
- 3 - ~ ~3~987 z~
layer, suitable examples being stated to include silver,
copper and zinc derivatives of sodium alginate.
Additionally, US Patent No. 4753231 discloses
the incorporation of such antibacterial agents as silver
sulphadiazine, povidone-iodine, chlorhexidine salts such
as gluconate, acetate and hydrochloride, and quaternary
agents such as benzalkonium chloride into a wound
dressing pad.
Moreover, US Patent No. 4817594 discloses wound
dressings as supports for therapeutic or antiseptic
materials including iodine, formol, lime, oxygen,
bacillary toxins and the like.
The commercially available products Bactigras
(trade mark) and Serotulle (trade mark), obtainable
respectively from Smith and Nephew Ltd., Hull, United
Kingdom, and Seton Healthcare Group, Oldham, United
Kingdom, both consist of paraffin ~auze impregnated with
the antibacterial agent chlorhexidine.
The medicated wound dressings of the prior art
comprise medicaments absorbed onto the surface of the
fibres making up the wound dressing. Such surface-bound
medicaments are expected to be released rapidly in the
environment of use. For a variety of clinical
applications, it is considered desirable to provide a
controlled release of the same or a different medicament
to a wound over time. If the medicament were merely
applied topically throughout the treatment regimen, this
would require frequent removal and replacement of the
dressing, with concomitant inconvenience and risk of
opportunistic infection.
We have now found that a medicament-çontaining
alginate fibre can be prepared which allows for the
sustained release of the medicament over time.
WO94/00164 PCT/GB93/01350
213~98~ ~-
- 4 -
The present invention accordingly provides an
alginate fibre comprising one or more medicaments
incorporated into the fibre core such that the or each
medicament can be released in a sustained manner over
time.
As used herein, "incorporated into the fibre
core" means encased within the fibre and is to be
contrasted with the situation where medicament is
attached to the surface of the fibre.
The time taken for substantially all of the
medicament or medicaments incorporated into the fibre
core to be released will depend on such factors as the
nature of the medicament(s) used, the amount of
medicament(s) used, and the permeability of the alginate
fibre. Different release periods will be required for
different clinical situations. Typically the or each
medicament will be released into the environment of use
over a period of hours, days or weeks. For example,
where the fibres of the invention are being used in a
wound dressing, the time taken for complete release of
medicament incorporated into the core of an alginate
fibre might be in the range of 1-7 days, for example 4-5
days.
It may frequently be desirable in the treatment
or management of wounds to provide for the possibility of
administering a medicament to a wound in a rapid initial
burst, followed by a slower controlled release of the
same or a different medicament to the wound. By way of
example, an antibacterial or antiseptic agent such as
chlorhexidine is generally applied promptly to a wound in
appreciable quantities so as to achieve a rapid initial
kill of deleterious micro-organisms, but this is
desirably followed up with further quantities of the
WO 94/00164 PCI/GB93/01350
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~ 213~987
medicament in smaller amounts over a sustained period in
order to prevent or inhibit bacterial Le~L~wth.
Accordingly, in a further aspect, the present
invention provides an alginate fibre comprising one or
S more medicaments incorporated into the fibre core and one
or more medicaments attached to the surface of the fibre,
whereby the or each medicament attached to the surface of
the fibre can be released rapidly therefrom, whilst the
or each medicament incorporated into the fibre core is
released in a sustained manner over time.
The rate of release of a medicament attached to
the surface of the fibre will be determined by a number
of factors such as the nature of the medicament and the
nature of the environment of use. Typically
substantially all of the medicaments attached to the
fibre surface will be released over a period of minutes
or hours, usually in the region of 5-60 minutes.
Incorporation of one or more medicaments into
an alginate fibre can be accomplished by a modification
of well-known procedu.es for the preparation of alginate
fibres. Procedures for the preparation of alginate
fibres are described in, for example, British Patents
Nos. 567641, 568177, 571657 and 624987. Essentially
these procedures comprise extruding an aqueous solution
of a water soluble alginate into a bath containing a salt
of a metal which forms a water insoluble alginate, such
as, for example, calcium chloride, so as to form a
thread.
We have now found that, if one or more
medicaments is (are) included in the solution of water
soluble alginate, when the solution, known as the "dope",
is extruded or spun into a solution of ions which form an
insoluble alginate, a fibre is formed having the
medicament(s) encased in the fibre core.
WO94/00164 c~ ~3 ~g 8~ PCT/GB93/01350
The present invention accordingly provides a
process for the preparation of an alginate fibre
comprising one or more medicaments incorporated into the
fibre core, which process comprises spinning a solution
of an alginate together with one or more medicaments.
One or more medicaments may be attached to the
surface of the fibres according to the invention by
physical or chemical means. Typical examples of
attachment by physical means include hydrogen bonding,
adsorption and van der Waals interaction. Alternatively,
some kind of chemical interaction between alginate
molecule and medicament molecule may occur, resulting in
cross-linking between the alginate and medicament
moieties. A typical example of attachment by chemical
means is ionic interaction, such as salt formation. For
example, chlorhexidine gluconate, which is the gluconate
salt of a cationic bisbiguanide derivative, possesses an
appreciable potential positive charge and has been found
to interact significantly with the negatively-charged
alginate molecule.
As will be appreciated, where the alginate
fibre according to the present invention comprises one or
more medicaments incorporated into the fibre core and one
or more medicaments attached to the surface of the fibre,
the individual medicaments incorporated into the fibre
core and attached to the surface of the fibre may be the
same or different. Preferably, a single medicament will
be incorporated into the fibre core; and likewise a
single medicament will preferably be attached to the
surface of the fibre. In a particularly preferred
embodiment, a single medicament is both incorporated into
the fibre core and attached to the surface of the fibre,
the medicament at each location being identical.
WO94/00164 PCT/GB93/01350
213~987
.~_
There is in principle no restriction on the
types of medicament which can be employed in conjunction
with the alginate fibre according to the invention. It
will nevertheless be understood that the or each
medicament to be incorporated into the fibre core must be
compatible in the sense that it should not interfere with
the spinning process by which the basic alginate fibre is
produced. Similarly, it will be understood that the or
each medicament to be attached to the surface of the
fibre will be such as to be capable of attachment thereto
by one or other of the means of attachment indicated
above. Suitable medicaments for use in conjunction with
the alginate fibre according to the invention include
antibacterial agents, for example bisbiguanide
derivatives such as chlorhexidine, both in the free base
form and as the acetate, gluconate or hydrochloride
salts, tetracycline derivatives such as
chlorotetracycline; oxytetracycline and tetracycline
itself, and sulphonamide derivatives such as
sulphadiazine; antiprotozoal agents, for example
imidazole derivatives such as metronidazole; antifungal
agents such as chlorphenesin; phenothiazine derivatives
such as promethazine and chlorpromazine;.nucleosides such
as iodouridine; hormones such as noradrenalin; and anti-
inflammatory agents, for example steroid derivatives suchas hydrocortisone and prednisolone. Preferred
medicaments for use in conjunction with the alginate
fibre according to the invention include chlorhexidine,
chlortetracycline, promethazine, noradrenalin and
prednisolone. Particularly preferred are chlorohexidine
and chlorotetracycline, especially chlorhexidine, in both
the free base and salt forms, more especially
chlorhexidine acetate.
WO94/00164 PCT/GB93/01350
2134987 '-
Dep~n~i~g upon the type(s) of medicament
incorporated into the core and, optionally, attached to
the surface, the fibre according to the invention may
possess a variety of advantages. In particular, as
indicated previously, an antibacterial or antiseptic
agent such as chlorhexidine may advantageously be both
incorporated into the fibre core and attached to the
surface of the fibre; a burst of medicament from the
surface of the fibre will achieve a rapid initial kill of
bacteria, whilst a sustained "follow-up" of smaller
quantities of medicament from the fibre core will deal
with bacterial eyLowth. For more complex medical
conditions different medicaments may advantageously be
incorporated into the fibre core and attached to the
surface of the fibre. For example, the medicament
incorporated into the fibre core may be an antifungal
agent whereas the medicament attached to the surface of
the fibre may be an antibacterial agent; or vice versa,
as n~cess~ry. Alternatively a binary system might be
envisaged, whereby the component released from the
surface of the fibre is initially in the form of an
inactive prodrug and only becomes activated, i.e.
converted into the active medicament, upon subsequent
release of the core component, with which the prodrug
interacts. By way of illustration, the medicament
incorporated into the fibre core might be an
antibacterial sulphonamide derivative such as
sulphadiazine, whilst the agent attached to the surface
of the fibre might comprise silver ions, which themselves
are known to pOCe~SS appreciable antibacterial activity;
rapid release of the silver ions into the medium would be
followed by a slower release from the core of
sulphadiazine, which would interact with the silver ions
and give rise to a product exerting a synergistically
WO94/00164 PCT/GB93/01350
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'~ 21~4987
enhanced antibacterial effect relative to that exerted by
either component alone.
The amount of medicament incorporated into the
fibre core will in general depend upon such factors as
S the solubility and stability of a given medicament in the
alginate dope from which the basic fibre is to be spun.
It may also depend upon the reactivity of a given
medicament with the alginate dope, since this may result
in an unspinnable mixture. ~or example, chlorhexidine
hydrochloride has been found to be relatively insoluble
in the alginate dope, resulting in a core concentration
of medicament of 0.2% w/w at most. Similarly, non-polar
species such as steroids are also relatively insoluble in
the alginate dope and thereby give rise to fibres
possessing a core concentration of medicament of
approximately 0.1% w/w. Moreover, chlorhexidine
gluconate, which, as mentioned above, has been found to
cross-link extensively with the alginate molecule, gives
rise to unspinnable mixtures at medicament concentrations
of well below 1.0% w/w. On the other hand, chlorhexidine
acetate has been found to perform well in the presence of
the alginate dope, with the result that core
concentrations of medicament in the region of 1.0% w/w
are readily achievable. As a general rule, the
concentration of medicament within the fibre core is
suitably from 0.01% to 2.0% w/w, preferably from 0.5% to
1.5% w/w.
The amount of medicament attached to the
surface of the fibre will also be dependent upon various
factors. These include, for example, the ability of a
given medicament to become adsorbed to the surface of the
pre-formed alginate fibre, or to cross-link chemically
with the alginate structure. In general, the
concentration of medicament attached to the surface of
-- 10 --
the fibre according to the invention is suitably from
0.01% to 2.0~ w/w, preferably from 0.5% to 1.5% w/w.
In a further aspect, the present invention
provides a process for preparing an alginate fibre
comprising one or more medicaments incorporated into the-
fibre core and one or more medicaments attached to the
surface of the fibre, which process comprises the
following steps:
(1) spinning a solution of an alginate
together with one or more medicaments in order to produce
a fibre incorporating the or each medicament within its
core; and
(2) modifying the resulting fibre by
attachment of one or more medicaments to the surface
thereof.
Suitable alginates for use in the processes
according to the invention include both water-soluble and
water-insoluble alginates, but will most preferably be
water-soluble alginates. A particular water-soluble
alginate for use in the spinning procedure is sodium
alginate. Nevertheless, the sodium alginate may
advantageously contain up to 1.5% by weight of calcium
ions. ~YAmples of specific sodium alginate products of
use in the process according to the invention include
Manucol DM (trade mark) which is available from Kelco
International Limited, and Protan LF lO/60 (trade mark),
which is available from Protan Limited.
Depending upon the nature of the alginate
and/or medicament(s) employed, the spinning procedure may
be tailored as required. For example, if a highly water-
soluble medicament is employed, so-called solv~nt
suppression may be required to prevent the medicament
being leached from the fibre during subsequent
processing; this involves the addition of a water-
'_ .h
~A
WO94/00164 PCT/GB93/01350
'~ ~ 2134387
miscible organic solvent, such as acetone or isopropanol,to the medicament-containing alginate dope.
Attachment of one or more medicaments to the
surface of the pre-formed alginate fibre may conveniently
be accomplished by one or other of a variety of standard-
procedures. Which method is adopted will, for example,
be dependent upon the relative ability of a given
medicament to become adsorbed to the pre-formed alginate
fibre, or upon its capacity for chemically cross-linking
with the alginate structure. Again, the procedure can be
tailored as required depending upon the nature of the
alginate and/or medicament(s) employed.
For instance, the medicament-containing
alginate fibre obtained from step (1) of the above
decribed process may be immersed in a bath containing an
aqueous solution of a given medicament or medicaments;
this procedure will clearly be particularly amenable to
appreciably water-soluble medicaments such as
chlorhexidine. If n~cesCAry~ a water-miscible cosolvent
such as acetone may be employed in the bath. The fibre
may advantageously be dragged through the bath in order
to promote the required amount of adsorption and/or
cross-linking at an acceptable rate. The duration of
immersion of the fibre in the medicament solution may
dictate the amount of medicament which is ultimately
adsorbed and/or cross-linked; this suggests that a
measure of quality control can be exercised over the
production of the final fibre, permitting a range of
fibres to be prepared having a variety of concentrations
of medicament attached to the surface, and hence a
corresponding range of amounts of medicament capable of
being released rapidly therefrom.
Alternatively, a solution of the or each
medicament in a volatile solvent such as acetone may be
WO94/00164 PCT/GB93/01350
213~987
- 12 -
sprayed onto the pre-formed alginate fibre obtained from
step (1) of the above-described process; upon evaporation
of the volatile solvent, a finished fibre results to
which a certain amount of medicament adheres, for example
by adsorption and/or chemical cross-linking. As will be
appreciated, this procedure lends itself particularly
appealingly to relatively poorly water-soluble
medicaments such as metronidazole.
The permeability of the finished fibre will be
influenced by the molecular structure of the particular
alginate employed. Alginates are block copolymers
consisting of guluronate and mannuronate, i.e. sugar-
derived, residues. One factor influential in determining
permeability will be the particular block structure of
the actual polymer employed, i.e. the precise
distribution within the molecule of the blocks of
guluronate and mannuronate residues. A high proportion
of guluronate blocks is known to promote a good
propensity for chelation of calcium ions. This in turn
means that a high ratio of guluronate to mannuronate
blocks can lead to a fibre having a lower permeability,
whereas a low ratio of guluronate to mannuronate blocks
will afford a more permeable fibre.
Alginates which comprise a high proportion of
guluronate residues are known as high G alginates, and
alginates which comprise a low proportion of guluronate
residues are known as low G alginates. Both high G
alginates and low G alginates are suitable for use in the
present invention.
Control of the amount of medicament
incorporated into the fibre core, and hence of the rate
of release of medicament from the fibre core, is
achievable by varying the concentration of the solution
in the bath from which the fibre is spun.
~WO94/00164 PCT/GB93/013~0
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213~987
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Another factor influencing the amount of
medicament incorporated into the fibre core is the
solubility of the medicament in the spin bath. Certain
medicaments are highly pH-dependent, and it is therefore
advisable to maintain the solution within the spin bath
at a constant optimum pH. For example, in the case of
chlorhexidine the spin bath solution is ideally kept at a
pH below 6Ø
Conventional fibre spinning proc~cc~c usually
involve a drying stage, towards or at the end of the
procedure. Drying may suitably be effected in an acetone
bath, or by means of heat. In the latter case, a less
dense fibre results than if acetone drying is involved;
this difference may again afford a valuable means of
controlling the permeability of the fibre core, and hence
the rate of release of medicament therefrom. Losses of
medicament from the fibre core during the acetone drying
stage may be encountered. This effect may be controlled
by varying the pH of the acetone drying bath(s) as
required.
In another aspect, the present invention
provides an alginate fabric formed in whole or in part
from the alginate fibre according to the invention.
The alginate fabric in accordance with the
invention may, for example, be non-woven, woven or
knitted. Preferably, the fabric is non-woven, not only
from the st~n~point of ease of manufacture but also
because of the general dimensional stability of non-woven
fabrics, which are acknowledged not to stretch so easily
as, for example, knitted fabrics.
In the preparation of a non-woven fabric, a
cotton card may be used to form a web, which may then be
cross-lapped, for example with a Garnet Bywater cross-
lapper, and then needle punched in a Garnet Bywater
- 14 -
needle loom. In the preparation of a woven fabric, the
precursor alginate fibres may be carded and then spun
into a yarn, which can be woven in a conventional loom.
Alternatively, the fibres may be collected in a spinning
box, according to the method described in British Patent
No. 568177, and woven. In the preparation of a knitted
fabric, the fibres can be prepared as a continuous
filament yarn, again according to the method described in
British Patent No. 568177, which is then knitted on a
conventional knitting machine.
The present invention further provides a wound
dressing comprising an alginate fabric according to the
invention.
As used herein, the expression "wound dressing"
includes surgical dressings. The term "wound" includes
burn, scald, cut, sore, ulcer, blister, rash or any other
lesion or area of troubled skin.
When adapted for use in the treatment of burns
or scalds, the wound dressing of the invention may
advantageously contain, either incorporated into the core
of the fibres constituting the dressing or attached to
the surface thereof, or both, one or more antimicrobial
agents known to be of particular efficacy in preventing
or inhibiting infection at the burn or scald site. Such
antimicrobial agents suitably include metronidazole, and
sulphonamide derivatives.
In order to prevent or retard the biological
degradation of the alginate fibre constituents, the wound
dressing of the invention will advantageously incorporate
conventional preservatives, for example Metasol D3T
(trade mark of Merck), Parasept (trade mark for methyl
paraben) (Kaloma Chemical) or Bromopol (trade mark for 2-
bromo-2-nitro-1,3-propanediol) (Boots Ltd.).
The wound dressings encompassed by the
invention may comprise one or more of the wound dressing
WO94/001~ PCT/GB93/01350
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~' 2134987
- 15 -
components well known in the art. For example, the wound
dressing may comprise one or more adhesive layers. The
adhesive layers may in~ep~n~ently comprise a continuous
or non-continuous layer, typically of thermoplastic.
Each of these layers may be impervious to moisture or
vapour. In the alternative the layer may be
semipermeable. Suitable materials have a softening point
of 70 to 120~C and may be polyamides such as
polycaprolactam and other "nylons", and also
polycarbonates or polyurethanes. Such materials may have
a pore size of less than 20 ~m. They may be water vapour
transmissive (200 to 2000 g/m2/24 hr) at the st~n~rd
relative humidity of 100% and stAn~rd temperature of
37-C. The effective pore size may be less than 2 ~m.
lSThe wound dressing may also comprise one or
more absorbent layers. The absorbent layers may
independently include, in addition to the alginate fibres
of the invention, karaya gum, locust bean gum, guar gum,
sodium acrylate, polyvinyl alcohol, pectin, gelatin,
carboxymethylcellulose, high molecular weight carbowaxes,
carboxy polymethyl collagen, cotton and carbon.
The wound dressing may also comprise a separate
and discrete layer which faces the wound. This wound
facing layer may optionally be a non-adhering
semipermeable thermoplastic selected from the class
described above.
The size and shape of the wound dressing may be
defined as desired. Typically the wound dressing will be
approximately circular or rectangular and may have length
or width or diameter of from 1 to 200 cm.
The wound dressings formed from the alginate
fabric according to the present invention will
advantageously be conventional dressings well known in
the art. Examples of suitable dressings include
W094/001~ 213 ~ 9 8 7 PCT/GB93/01350
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bandages, adhesive strip dressings, island dressings,
pads of various kinds, surgical sponges and packs, ward
dressings of the type sold under the registered Trade
Mark "Steripad", and such articles as tampons which may,
for example, be impregnated with an antifungal agent such
as miconazole for the treatment of candidal vaginitis
(vaginal thrush). Such dressings may conveniently be
prepared by stAn~Ard methods known from the art.
The dressings in accordance with the present
invention will conveniently be packaged in a
hermetically-sealed envelope and sterilised, e.g. with
ethylene oxide or by gamma-irradiation.
In a still further aspect, the present
invention provides a method of treating a wound which
comprises the application to a wound of a wound dressing
according to the invention.
Embodiments of the invention will now be
described by way of example and with reference to the
accompanying drawings, in which:
Figure 1 is a schematic representation of a
suitable apparatus for preparing alginate fibre
comprising medicament incorporated into the fibre core;
Figure 2 is a plot of percentage release of
chlorhexidine as a function of incubation time for an
alginate fibre comprising chlorhexidine incorporated into
the fibre core; and
Figure 3 is a plot of the ~once~tration of
chlorhexidine in mg per gram of fibre attached to the
surface of alginate fibre as a function of the duration
of immersion of the unloaded fibre in a chlorhexidine
bath.
WO94/00164 PCT/GB93/01350
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- 17 -
Method A
Basic Method for Preparation of Alginate Fibres
containinq Chlorhexidine
Alginate dope containing chlorhexidine was
prepared by dissolving sodium alginate (5 g) in deionised
water (95 ml) containing the appropriate amount of
chlorhexidine diacetate (0.05 g). The mixture was
stirred using a Silverson mixer until obvious particulate
matter had been dispersed. The mixture was then allowed
to stand for 24 hours. Using the apparatus depicted
schematically in Figure 1, the mixture was extruded, by
means of pump 1, through a 400 hole spinneret 2 into a
bath 3 containing a solution of calcium chloride and
sodium chloride. The rate of extrusion could be
controlled by means of the pump speed controller 4 in
conjunction with pressure gauge 5. The resulting fibre
was stretched between two godets 6 and 7, whose operation
was controlled by godet controllers 8 and 9 respectively.
The fibre was then collected on a wind-up spool 10,
itself controlled by controller 11. The direction of
passage of the nascent fibre through the.apparatus was
controlled by fibre guides 12, 13 and 14. The collected
fibre was then dried using acetone, and the acetone
driven off in a stream of warm air. Yield, 4.5 g fibre;
chlorhexidine diacetate content 0.5% w/w.
WO94/00164 PCT/GB93/01350
21349~7 ~
- 18 -
Method B
Anal~tical Method for Determination of Chlorhexidine
Diacetate Content in Fibres
3.0 mg of chlorhexidine diacetate was dissolved
in 20 ml of 2N hydrochloric acid and then treated as
described below. By a process of dilution, solutions
containing 2.4 mg, 1.8 mg, 1.2 mg and 0.6 mg of
chlorhexidine diacetate were prepared and treated in
similar fashion. Absorptions were determined at 475 nm
and a calibration curve prepared.
400 mg of the fibre, prepared as described in
Method A above, was weighed out, and placed in a small
beaker. The fibre was cut into small pieces to ensure
complete extraction of the chlorhexidine. 20 ml of 2N
hydrochloric acid was added, and the mixture agitated.
This was then filtered under vacuum. The filtrate was
placed in a 100 ml volumetric flask. The volume was
adjusted to about 80 ml with deionised water. 5.0 ml of
cetrimide solution (prepared by dissolving 20 g of
cetrimide B.P. in 80 ml of warm deionised water; cooling;
and diluting to 100 ml) was added, then 2.0 ml of
alkaline sodium hypobromite, prepared as described below.
If ne~CF~ry~ small amounts of sodium hydroxide were
added until the solution was alkaline; this point was
detected by a colour change in the solution from yellow
to orange. The solution was then made up to 100 ml using
deionised water. Absorption was then determined at 475
nanometers, and the chlorhexidine diacetate content of
the fibre could thence be calculated with reference to
the calibration curve obtained as described above.
WO94/001~ PCT/GB93/01350
213~987
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-- 19 --
The alkaline sodium hypobromite reagent
utilised in the above-described procedure can be prepared
as follows:
Place 5 ml of bromine in a measuring cylinder.
Add to 400 ml of sodium hydroxide solution, which has
been prepared by adding 10 g of sodium hydroxide to 400
ml of deionised water in a concial flask. Stir until all
the bromine has gone into solution. Adjust the volume of
this solution to 500 ml with deionised water. The
working reagent is prepared by diluting 100 ml of the
resulting solution to 150 ml with deionised water. Place
132 ml in a measuring cylinder, and add 66 ml of 3N
sodium hydroxide. This is stable for about one month.
Method C
Release of Chlorhexidine from Fibres
Fibre, prepared as described in Method A above,
was cut up into twenty-four 100 mg samples as follows:
A length of fibre was cut from the main body of
the fibre, i.e. from one particular area. This was then
cut up into three 100 mg samples of equal lengths
(labelled a, b and c respectively), keeping a further 100
mg off-cut to be used as the control sample for each
batch ~labelled Control). Each 100 mg sample was then
placed in small, individual sample vials, labelled
accordingly. Samples a, b and c had 2 ml of a pure
culture of Pseudomonas aeru~inosa added to them, ensuring
all the fibre was covered. The Control sample for each
batch was left unaltered. All samples were incubated at
37~C for 75 min, and the above procedure was repeated for
corresponding samples for other specific periods of time
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- 150 min, 225 min, 300 min, 375 min and 450 min -
reguiring twenty-four samples in all.
The samples were then analysed for
chlorhexidine content as described in Method B above. A
mean value of the individual a, b and c readings for each
incubation period was calculated, and the results
obtained are displayed in Table 1 below and also depicted
in Figure 2, which plots the percentage of chlorhexidine
released against time (as used herein, CX is an
abbreviation for chlorhexidine).
TABLE 1
Incubation Time (minutes) Amount of CX Released (%)
150 27
225 24
300 31
375 30
450 38
From Table 1 and Figure 2, it can be seen that
a steady sustained release of chlorhexidine from the
fibre core occurs with time.
Method D
Pre~aration of Alginate Fibre with Chlorhexidine
Diacetate on the Fibre Surface
3 1 of a saturated solution of chlorhexidine
diacetate in deionised water was prepared. Approximately
8 cm lengths of calcium alginate fibre tow were immersed
in this solution for known lengths of time. The samples
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were then dried by brief immersion in acetone, the
acetone subsequently being driven off in a stream of warm
air. The fibre samples were then analysed as described
in Method B above. The results obtained are displayed in
Table 3 below and also depicted in Figure 3, which plots-
the concentration of chlorhexidine diacetate attached to
the fibre surface as a function of the immersion time.
TABLE 3
Immersion Concentration of Chlorhexidine
time (sec~ diacetate on fibre surface (mq/a fibre)
0.9575
1.0925
1.4475
1.8300
1.8725
120 8.8300
180 8.7525
300 13.6525
As will be seen from Figure 3, the
concentration of chlorhexidine which becomes attached to
the fibre surface is essentially proportional to the
duration of immersion of the fibre in the chlorhexidine
diacetate solution bath.
Method E
Experiment to Determine Viability of Chlorhexidine after
Attachment to Surface of Alainate Fibre
A non-irradiated sample of fibre which had been
treated with chlorhexidine on its surface, as described
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in Method E above, was used to determine the range of
bacterial susceptibility. The following bacterial
strains were selected for te~ting:
Candida albicans NCPF 3255
Pseudomonas aeruginosa NCTC 10322
Escherischia coli NCTC 9001
Proteus vulgaris NCTC 4175
Four 10 ml samples of tryptone soya broth were
inoculated with each strain and a plate count done on
each. Then a 100 mg sample of the fibre was added to
half the broths and the remainder left as controls.
Those broths to which fibre had been added were then
sampled again and then at hourly intervals, by removing
0.1 ml aliquots plated out onto nutrient agar, the
control broths being sampled only at the beginning and
the end of the experiment. The results obtained are
displayed in Table 4 below.
TABLE 4
A. 100 mg of fibre in 10 ml of ;nor~lated broth
Time Ps Ps(D) Pr Pr(D) Can Can(D) Esc Esc(D)
(mins)
o U/C U/C 38 32 U/C U/C U/C U/C
5 U/C U/C 70 60 U/C U/C U/C U/C
24 0 0 6 1 0 0
120 21 50 0 0 1 0 0 0
180 6 1 0 0 o 0 0 o
240 0 o o o o o o o
300 0 8 0 0 0 0 0 0
360 0 4 0 0 0 0 0 0
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B. Control broth s~mples
Time Ps Ps(D) Pr Pr(D) Can Can(D) Esc Esc(D)
(mins)
o U/C U/C 52 46 U/C U/C U/C U/C
360 C C U/C U/C U/C C C C
U/C = an uncountable number of colonies
C = confluent growth
Can = Candida albicans NCPF 3255
Ps = Pseudomonas aeruginosa NCTC 10322
Esc = Escherischia coli NCTC 9001
Pr = Proteus vulgaris NCTC 4175
(D) = duplicate measurement
From the results displayed in Table 4 above it
is apparent that chlorhexidine is not inactivated by
attachment to alginate fibres, since all bacterial
strains tested showed a high degree of sensitivity to
chlorhexidine released from the fibre surface. This
compound was demonstrably active within the first hour of
incubation, and remained active throughout the six hours
of testing. These results apparently contradict various
reports in the literature, which suggest that
chlorhexidine becomes deactivated upon contact with
alginate.
